In general, the present invention relates to methods for diagnosing or treating a disease, as well as for identifying a subject for participation in a clinical trial, and identifying a subject at risk for a disease.
Many diseases are difficult to diagnose because the appropriate diagnostic tools have not yet been identified. The ability to predict that an a symptomatic subject is at risk for developing a disease is even more difficult. A method that would provide a better means by which a disease could be diagnosed, or the risk of developing a disease could be assessed, would be beneficial. The result of having better diagnostic or risk assessment tools would be the more timely administration of appropriate therapies. In addition, not all patients with the same disease respond with equal efficacy to the same therapy. The genotype of a patient may affect the pharmacological efficiency among patients having the same disease. If the genotype of a diseased patient is known, optimal therapies can be determined and administered for the patient, resulting in a faster recovery from the disease.
The present invention provides methods for diagnosing or treating a disease, as well as for identifying a subject for participation in a clinical trial, and for identifying a subject at risk for a disease. The methods of the invention involve genotyping or phenotyping subjects for the presence of a variant PON1 allele. The information obtained from the determination of the PON1 allele status can be used to diagnose the subject as having a disease, or to identify the subject as being at risk for a disease, or to determine the appropriate therapy for the subject. PON1 allele status determination can also be helpful in designing and assessing the results of a clinical trial aimed at developing a therapy for the treatment of a disease. In a related aspect, the invention features a treatment protocol that provides a prediction of patient outcome.
The human PON1 gene, as reviewed by Mackness et al. (Gen. Pharmac. 31:329-336, 1998), encodes a serum paraoxonase protein. The protein is a 45-kDa glycoprotein that is associated with high density lipoprotein. The protein functions by hydrolyzing organophosphate insecticides and nerve gases, and is responsible for determining the selective toxicity of these compounds in mammals.
Historically, the amino acid residues of the paraoxonase protein may be numbered in two different ways. The amino acids of paraoxonase may be numbered with methionine (beginning at base pair 10) or with alanine (beginning at base pair 13; as used herein) as the first amino acid (see FIG. 1; SEQ ID NO:1). A variant PON1 allele may occur, for example, at amino acid position 54, as used herein, or 55, depending on which numbering system is used. For this reason, the mutation Met54Leu is equal to the mutation Met55Leu. For clarity, this application utilizes the numbering system beginning with alanine as the first amino acid, and therefore refers to PON1 alleles at amino acid positions 54 and 191.
Accordingly, in one aspect, the present invention features a method for identifying a subject at risk for a disease. The method includes genotyping or phenotyping the PON1 locus of a subject, and determining the presence of a variant PON1 allele or isoform. The presence of such a variant allele or isoform indicates an increased risk for the disease.
In a second aspect, the present invention features a method for diagnosing a subject with a disease. The method includes genotyping or phenotyping the PON1 locus of a subject, and determining the presence of a variant PON1 allele or isoform. The presence of such a variant allele or isoform indicates an increased risk for the disease.
In a third aspect, the present invention features a method for identifying a subject for participation in a clinical trial of a therapy for the treatment of a disease. The method includes genotyping or phenotyping the PON1 locus of a subject, and determining the presence of a variant PON1 allele or isoform, where the presence of a variant PON1 allele or isoform places the subject into a subgroup for a clinical trial of a drug.
In a fourth aspect, the present invention features a method of treating a subject with a disease. The method includes genotyping or phenotyping the PON1 locus of a subject, determining the presence of a variant PON1 allele or isoform, and determining the preferred therapy for the treatment of the disease.
In preferred embodiments of all of the above aspects of the invention, the disease may be a neurological disease. The neurological disease can be Alzheimer""s disease (AD), or a non-Alzheimer""s disease neurological disease (non-AD). In a preferred embodiment, the neurological disease is Alzheimer""s disease, neurofibromatosis, Huntington""s disease, depression, amyotrophic lateral sclerosis, multiple sclerosis, stroke, Parkinson""s disease, or multi-infarct dementia. In further embodiments of the above aspects of the invention, the variant PON1 allele or isoform contains a deletion, insertion, or missense mutation. In another preferred embodiment, the PON1 allele status is heterozygous or homozygous for the PON1 Met54Leu allele.
In preferred embodiments of the invention, the therapy can be a cholinomimetic therapy (e.g. tacrine) or a non-cholinomimetic therapy (e.g., a vasopressinergic therapy). In another preferred embodiment, the therapy can be probucol, a monoamine oxidase inhibitor, a muscarinic agonist, a neurotrophic factor, a noradrenergic factor, an antioxidant, an anti-inflammatory agent, corticotrophin-releasing hormone (CRH), somatostatin, substance P, neuropeptide Y, and thyrotrophin-releasing hormone (TRH).
In a particular application of the invention, all of the above aspects feature a determination of the PON1 allele status of the subject, where a determination of the PON1 allele status, e.g., of the Met54Leu variant, as being heterozygous or homozygous, is predictive of the patient having a poor response to a therapy for a neurological disease (e.g., Alzheimer""s disease).
The invention also provides a method for treating a patient at risk for a disease by a) identifying a patient with a risk, b) determining the PON1 allele status of the patient, and c) converting the data obtained in step b) into a treatment protocol that includes a comparison of the PON1 allele status with the allele frequency of a control population. This comparison allows for a statistical calculation of the patient""s risk for having a particular disease. In preferred embodiments, the method provides a treatment protocol that predicts a patient being heterozygous or homozygous for the Met54Leu allele to respond poorly to a cholinomimetic (e.g., tacrine) or specific non-cholinomimetic (e.g., vasopressinergics) therapy for a neurological disease, and a patient who is wild type homozygous, to respond favorably to the therapy.
The invention also provides treating a patient at risk for, or diagnosed with, a disease using the above method, and conducting an additional step c) which involves determining the apolipoprotein E allele (e.g., apoE4) or butyrylcholinesterase allele (e.g., BCHE-K) load status of the patient. This method further involves converting the data obtained in steps b) and c) into a treatment protocol that includes a comparison of the allele status of these steps with the allele frequency of a control population. This affords a statistical calculation of the patient""s risk for having a disease, for example, a neurological disease. In a preferred embodiment, the method is useful for treating a neurological disease such as Alzheimer""s disease, neurofibromatosis, Huntington""s disease, depression, amyotrophic lateral sclerosis, multiple sclerosis, stroke, Parkinson""s disease, or multi-infarct dementia. In addition, in related embodiments, the methods provide a treatment protocol that predicts a patient to be at high risk for a neurological disease and responding poorly to a cholinomimetic or particular non-cholinomimetic therapy (e.g., vasopressinergics) if the patient is determined to have all or any combination of a PON1 Met54Leu allele, an apoE4 allele, and a BCHE-K allele. Such patients are preferably given an alternative therapy.
The treatment protocol can include a therapy plan for a patient using genetic and diagnostic data, including the patient""s neurological diagnosis and PON1, BCHE, and ApoE genotypes. The protocol enhances therapeutic options and clarifies prognoses. The treatment protocol may include an indication of whether or not the patient is likely to respond positively to a cholinomimetic or non-cholinomimetic therapy. The treatment protocol may also include an indication of appropriate drug dose, recovery time, age of disease onset, rehabilitation time, symptomology of attacks, and risk for future disease. A treatment protocol, including any of the above aspects, may also be formulated for a symptomatic and healthy subjects in order to forecast future disease risks and determine what preventive therapies should be considered or invoked in order to decrease these disease risks. The treatment protocol may include the use of a computer software program to analyze patient data.
Genotype determinations can be compiled to predict either prognosis, drug efficacy, or suitability of a patient for participating in clinical trials of a neurological disease therapeutic. For example, the genotype may be compiled with other patient parameters such as age, sex, disease diagnosis, and known allelic frequency of a representative control population. A determination of the statistical probability of the patient having a particular disease risk, drug response, or patient outcome may be assessed from such genotype determinations. Patient outcome, i.e. a prediction of a patient""s likely health status, may include a prediction of the patient""s response to therapy, rehabilitation time, recovery time, cure rate, rate of disease progression, predisposition for future disease, or risk of having relapse.
The invention also provides a method for improving the efficacy of a therapy for the treatment of diseases. The method includes the step of comparing the relative efficacy of the therapy in patients having different PON1 alleles. Preferably, administration of the drug is preferentially provided to those patients with a PON1 allele type associated with increased efficacy. In a preferred embodiment, the alleles of PON1 used are wild type PON1 and PON1 associated with reduced biological activity. Most preferably the allele associated with reduced biological activity is PON1 Met54Leu.
By xe2x80x9cdiseasexe2x80x9d is meant a condition of a living animal that impairs the normal performance or function of the animal.
As used herein, by xe2x80x9cgenotypingxe2x80x9d is meant determination of the type and number of alleles present in a subject, whether determined by nucleic acid sequencing, PCR or RT-PCR amplification, examination of PON1 protein, or by other methods available to those skilled in the art. A specific gene can be genotyped to determine if the gene is a wild-type or variant allele.
By xe2x80x9cphenotypingxe2x80x9d is meant to determine the detectable outward manifestations of a genotype. For example, the detection of a polypeptide using an epitope-specific antibody is one method of phenotyping.
By xe2x80x9cPON1 genexe2x80x9d is meant a gene encoding the paraoxonase polypeptide. In one embodiment, the PON1 gene is human.
By xe2x80x9cparaoxonase proteinxe2x80x9d or xe2x80x9cparaoxonase polypeptidexe2x80x9d is meant a polypeptide or fragment thereof, encoded by the PON1 gene. In one embodiment, the paraoxonase protein or polypeptide is human.
By xe2x80x9cPON1 allele statusxe2x80x9d is meant a determination of the relative ratio of wild type paraoxonase alleles compared to an allelic variant that may encode a paraoxonase gene product of reduced catalytic activity. This may be accomplished by nucleic acid sequencing, RT-PCR, PCR, examination of the paraoxonase protein, a determination of the paraoxonase enzyme activity, or by other methods available to those skilled in the art.
By xe2x80x9cwild-typexe2x80x9d is meant any allele, or polypeptide encoded by such an allele, that is present in that part of the population considered free of the particular disease for which the variant allele is being assessed for association with prognosis, diagnosis, or therapeutic efficacy.
By xe2x80x9cvariant PON1 allelexe2x80x9d is meant any sequence mutation of the paraoxonase (PON1) gene that differs from the predominant wild-type allelic sequence (e.g., a variant PON1 allele that changes amino acid residue 54 from leucine to methionine) and which may result in abnormal paraoxonase activity (including protein levels). A variant PON1 allele not specifically described to be associated with a disease herein can be tested for association using the techniques provided herein and those known in the art.
By xe2x80x9cparaoxonase activityxe2x80x9d is meant the function of the paraoxonase protein. Function includes, but is not limited to, protein level, degree of enzymatic function, and interactions of paraoxonase with other molecules, including, but not limited to polypeptides and nucleic acids.
By xe2x80x9ctreatingxe2x80x9d is meant the medical management of a subject, e.g., a human patient, with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder. This term includes active treatment, that is, treatment directed specifically toward the improvement or associated with the cure of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder. In addition, this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder. xe2x80x9cTreatingxe2x80x9d also includes symptomatic treatment, that is, treatment directed toward constitutional symptoms of the associated disease, pathological condition, or disorder. Thus xe2x80x9ctreatingxe2x80x9d includes submitting or subjecting a subject to a compound which will promote the elimination or reduction of a disease or symptoms of a disease, or which will slow the progression of said disease. For example, a subject may be treated with, synthesized organic molecules, naturally occurring organic molecules, peptides, polypeptides, nucleic acid molecules, and components thereof xe2x80x9cTreatingxe2x80x9d also includes the act of not giving a subject a contra-indicated therapeutic.
As used herein, by xe2x80x9cdeletion mutationxe2x80x9d is meant a mutation in a gene resulting from the absence of at least one nucleotide, when compared to the wild- type nucleotide sequence. A polypeptide encoded by a deletion mutation gene may contain an altered amino acid sequence. The polypeptide may be shortened, lengthened, or contain different amino acids as compared to the polypeptide encoded by the wild-type gene.
By xe2x80x9cinsertion mutationxe2x80x9d is meant a mutation in a gene resulting from the insertion of at least one nucleotide, when compared to the wild-type nucleotide sequence. A polypeptide encoded by an insertion mutation gene may contain an altered amino acid sequence. The polypeptide may be shortened, lengthened, or contain different amino acids as compared to the polypeptide encoded by the wild-type gene.
By xe2x80x9cmissense mutationxe2x80x9d is meant a mutation in a gene resulting from the substitution of at least one nucleotide in the sequence for another, when compared to the wild-type nucleotide sequence. A polypeptide encoded by a missense mutation gene may contain an altered amino acid sequence. The polypeptide may be shortened, lengthened, or contain different amino acids as compared to the polypeptide encoded by the wild-type gene.
By xe2x80x9chomozygous for the PON1 Met54Leu allelexe2x80x9d is meant that both copies of the PON1 allele in a cell are identical, and encode a methionine at amino acid position 54 of the paraoxonase polypeptide.
By xe2x80x9cheterozygous for the PON1 Met54Leu allelexe2x80x9d is meant that the two copies of the PON 1 allele in a cell are not identical. For example a cell which is heterozygous for the PON1 Met54Leu allele contains one allele which codes for a methionine at amino acid position 54 of the paraoxonase polypeptide, while the other allele codes for a leucine at the same amino acid position.
By xe2x80x9cneurological diseasexe2x80x9d is meant a disease which involves the neuronal cells of the nervous system. Specifically included are: prior diseases (e.g., Creutzfeldt-Jakob disease); pathologies of the developing brain (e.g., congenital defects in amino acid metabolism, such as argininosuccinicaciduria, cystathioninuria, histidinemia, homocystinuria, hyperammonemia, phenylketonuria, tyrosinemia, and fragile X syndrome); pathologies of the mature brain (e.g., neurofibromatosis, Huntington""s disease, depression, amyotrophic lateral sclerosis, multiple sclerosis); conditions that strike in adulthood (e.g., Alzheimer""s disease, Creutzfeldt-Jakob disease, Lewy body disease, Parkinson""s disease, Pick""s disease); and other pathologies of the brain (e.g., brain mishaps, brain injury, coma, infections by various agents, dietary deficiencies, stroke, multiple infarct dementia, and cardiovascular accidents).
By xe2x80x9cAlzheimer""s diseasexe2x80x9d or xe2x80x9cADxe2x80x9d is meant a pathology characterized by an early and extensive loss of entorhinal cortex neurons. Alzheimer""s disease subjects may be identified by progressive and degenerative effects on the brain which are not attributable to other causes. A diagnosis of Alzheimer""s disease is made using clinical-neuropathological correlations known in the art (see e.g., Arch. Neurology 51:888-896, 1994). Post-mortem, the disease may be diagnosed by the presence of amyloid plaques and fibrils.
By xe2x80x9cnon-AD neurological diseasexe2x80x9d is meant a disease other than Alzheimer""s disease, which involves the neuronal cells of the nervous system. Specifically included are: prior diseases (e.g, Creutzfeldt-Jakob disease); pathologies of the developing brain (e.g., congenital defects in amino acid metabolism, such as argininosuccinicaciduria, cystathioninuria, histidinemia, homocystinuria, hyperammonemia, phenylketonuria, tyrosinemia, and fragile X syndrome); pathologies of the mature brain (e.g., neurofibromatosis, Huntington""s disease, depression, amyotrophic lateral sclerosis, multiple sclerosis); conditions that strike in adulthood (e.g. Creutzfeldt-Jakob disease, Lewy body disease, Parkinson""s disease, Pick""s disease); and other pathologies of the brain (e.g., brain mishaps, brain injury, coma, infections by various agents, dietary deficiencies, stroke, multi-infarct dementia, and cardiovascular accidents).
As used herein, by xe2x80x9ctherapyxe2x80x9d is meant any treatment suitable for treating a disease. The purpose of the therapy is to reduce or eliminate the disease, or a symptom associated with the disease, or to inhibit the disease from progressing further. In addition, the term therapy may also include the close monitoring of an a symptomatic subject for the appearance of any symptoms of a disease. xe2x80x9cTherapy for the treatment of a neurological diseasexe2x80x9d is any therapy suitable for treating a neurological disease. A suitable therapy can be a pharmacological agent or drug that may enhance cognitive function, motor function, or neuronal activity of the central nervous system, peripheral nervous system, or inhibit the further deterioration of any of these faculties. Drug efficacy of an appropriate drug can be determined by drug dosage, administration schedule, and prediction of therapeutic utility.
By xe2x80x9ccholinomimetic therapyxe2x80x9d is meant any drug that mimics the function of acetylcholine or enhances the activity of acetylcholine synthesizing cells. These drugs include, but are not limited to, inhibitors of acetylcholine degradation (acetylcholine esterase inhibitors such as tacrine), drugs that mimic acetylcholine structure and function, drugs that block acetylcholine uptake by neurons, and drugs that interact with pre-synaptic receptors to induce acetylcholine release from cholinergic neurons.
By xe2x80x9cnon-cholinomimetic therapyxe2x80x9d is meant a therapy that, for example, utilizes a vasopressinergic modulator.
By xe2x80x9cdetermining the presence of a variant PON1 allelexe2x80x9d is meant subjecting a nucleic acid sample to any of a variety of detection techniques know in the art for elucidating a mutation in a nucleic acid (e.g., polymerase chain reaction (PCR), reverse transcriptase-PCR (RT-PCR), ligase-mediated chain reaction step, chip hybridization methods, sequencing nucleic acids by mass spectrometry, or restriction enzyme-mediated digestion). The mutation may be an insertion, deletion, or missense mutation. For example, in the presence of appropriately designed primers, a nucleic acid fragment can be amplified using PCR, and analyzed by restriction enzyme digestion that can reveal the presence of a variant allelic sequence. In addition, DNA sequencing may be employed using techniques known in the art. These nucleic acid techniques allow for a genotype determination of the PON1 locus.
Alternatively, phenotyping of the PON1 locus may be performed (and a genotype thus inferred) by using standard techniques for detecting the presence of a polypeptide having a particular amino acid change (e.g., antibodies, isoelectric focusing, and 2-D PAGE). For example, the presence of a variant paraoxonase polypeptide (e.g., Met54Leu) can be distinguished from a wild-type paraoxonase polypeptide using epitope-specific antibodies available in the art. Epitope-specific antibodies can be generated using the methods of Harlow and Lane (Antibodies. A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 1988). The sequence of the antigen used to generate antibodies capable of detecting variant PON1 alleles would include the mutated amino acid sites encoded by the variant alleles.
By xe2x80x9crisk factor associated with a diseasexe2x80x9d is meant any risk factor for a disease known in the art. Examples of risk factors commonly associated with diseases include age, gender, diet, exercise, weight, the presence of another disease, and the occurrence of a specific genotype. Risk factors associated with a neurological disease in particular may include advanced age, lower intelligence, smaller head size, history of head trauma, mutations on chromosomes 1, 14, and 21, presence of an apoE4 allele, or the presence of a BCHE-K allele (see e.g., Cummings et al., Neurology (1 Supp.1):S2-S17, 1998).
By xe2x80x9csubject at risk for a diseasexe2x80x9d is meant a subject identified or diagnosed as having a disease or having a genetic predisposition or risk for acquiring a disease using the methods of the invention and techniques available to those skilled in the art.
The present invention provides a number of advantages. For example, the methods described herein allow for the determination of a subject""s PON1 genotype for the timely administration of an optimal treatment for the disease. The invention also allows for the identification of a subject at risk for a disease so that a prophylactic therapy for the treatment of the disease may be started before symptoms of the disease are apparent.
Other features and advantages of the invention will be apparent from the following detailed description and from the claims.